Restricted Localization of the TNF Receptor CD120a to Lipid Rafts: a Novel Role for the Death Domain

Journal of Immunology (Baltimore, Md. : 1950). Apr, 2002  |  Pubmed ID: 11937569

The TNF-alpha receptor, CD120a, has recently been shown to be localized to both plasma membrane lipid rafts and to the trans Golgi complex. Through a combination of both confocal microscopy and sucrose density gradient ultracentrifugation, we show that amino acid sequences located within the death domain (DD) of CD120a are both necessary and sufficient to promote the appropriate localization of the receptor to lipid rafts. Deletion of the DD (CD120a.Delta321-425) prevented the receptor from being targeted to lipid rafts and resulted in a uniform plasma membrane localization. A similar loss of raft localization was also observed following pairwise deletion of the six alpha-helices that comprise the DD. In all situations, the loss of the ability of CD120a to become localized to lipid rafts following mutagenesis was paralleled by a failure of the receptor to initiate apoptosis. Furthermore, introduction of the lpr mutation into CD120a (CD120a.L351N) also resulted in both a loss in the ability of the receptor to signal apoptosis and to be appropriately localized to rafts. In contrast to CD120a, CD120b, which lacks a DD, is mainly expressed in the bulk plasma membrane and to a lesser extent in lipid rafts, but is absent from the Golgi complex. However, a chimeric receptor in which the DD of CD120a was fused to the cytoplasmic domain of CD120b was predominantly localized to lipid rafts. Collectively, these findings suggest that in addition to its role in CD120a signaling, an appropriately folded and functionally active DD is required for the localization of the receptor to lipid rafts.

Impairment of Macrophage Survival by NaCl: Implications for Early Pulmonary Inflammation in Cystic Fibrosis

American Journal of Physiology. Lung Cellular and Molecular Physiology. Jul, 2002  |  Pubmed ID: 12060576

Inflammation, characterized by the presence of proinflammatory chemokines and neutrophils, is a hallmark of early airway disease in infants with cystic fibrosis (CF), although the underlying mechanisms remain poorly defined. In this study, we evaluated the role of NaCl and the ensuing hyperosmolar effect on tumor necrosis factor (TNF)-alpha signaling and apoptosis in macrophages. Incubation of mouse macrophages with NaCl activated p38(mapk) and the p46(jnk) and p54(jnk) c-jun NH(2)-terminal kinase isoforms, but not p42(mapk/erk2) or Akt. Similar results were obtained with sorbitol, suggesting a general response to hyperosmolarity. Strikingly, the activation of p42(mapk/erk2) and Akt by TNF-alpha was also inhibited in the presence of NaCl. Because the activation of p42(mapk/erk2) and Akt has been associated with survival responses, we investigated the effect of NaCl on macrophage apoptosis. The results indicated a synergistic increase in apoptosis when macrophages were exposed to TNF-alpha in the presence of NaCl compared with stimulation with TNF-alpha alone or NaCl alone. Furthermore, pharmacological inhibition of p42(mapk/erk2) and Akt mimicked the effect of NaCl. Collectively, these findings indicate that modest elevations in NaCl differentially regulate the activation of mitogen-activated protein kinases and Akt and potentiate macrophage apoptosis. We speculate that augmentation of macrophage apoptosis in CF airways may result in decreased clearance of neutrophils and in deficiencies in the elimination of common CF pathogens.

Induction of Macrophage Insulin-like Growth Factor-I Expression by the Th2 Cytokines IL-4 and IL-13

Journal of Immunology (Baltimore, Md. : 1950). Oct, 2003  |  Pubmed ID: 14500651

Macrophage-derived insulin-like growth factor-I (IGF-I) has long been implicated in the pathogenesis of the interstitial lung disease, idiopathic pulmonary fibrosis, in part, by its ability to 1) stimulate the proliferation and survival of fibroblasts and myofibroblasts and 2) promote collagen matrix synthesis by these cells. However, little is known about the mechanisms that stimulate the expression of IGF-I by macrophages. Previous studies have shown that the development of pulmonary fibrosis is accompanied by enhanced expression of Th2-profile cytokines, especially IL-4, and diminished expression of Th1 cytokines, including IFN-gamma. In addition, in vitro studies have shown that IFN-gamma down-regulates the expression of IGF-I. Thus, the paucity of IFN-gamma in the fibrotic lung may favor increased growth factor production by allowing Th2 cytokines to predominate. In view of these findings, we investigated the hypothesis that Th2 cytokines stimulate the expression of IGF-I by macrophages. Incubation with IL-4 or IL-13 led to concentration- and time-dependent increases in the expression of IGF-I mRNA and the secretion of IGF-I protein by mouse macrophages as a consequence of increased transcription of IGF-I pre-mRNA. Exposure of macrophages to IL-4 in the presence of IFN-gamma inhibited the increase in the expression of IGF-I. Studies using STAT6-deficient macrophages indicated that the increase in IGF-I expression was dependent on STAT6. In addition, the down-regulation of IGF-I expression by IFN-gamma was absent in STAT1-deficient macrophages. Collectively, these findings define a homeostatic mechanism in which Th2 cytokines promote, and Th1 cytokines inhibit, the expression of IGF-I by macrophages.

TRUSS, a Novel Tumor Necrosis Factor Receptor 1 Scaffolding Protein That Mediates Activation of the Transcription Factor NF-kappaB

Molecular and Cellular Biology. Nov, 2003  |  Pubmed ID: 14585990

We describe the cloning and characterization of tumor necrosis factor receptor (TNF-R)-associated ubiquitous scaffolding and signaling protein (TRUSS), a novel TNF-R1-interacting protein of 90.7 kDa. TRUSS mRNA was ubiquitously expressed in mouse tissues but was enriched in heart, liver, and testis. Co-immunoprecipitation experiments showed that TRUSS was constitutively associated with unligated TNF-R1 and that the complex was relatively insensitive to stimulation with TNF-alpha. Deletion mutagenesis of TNF-R1 indicated that TRUSS interacts with both the membrane-proximal region and the death domain of TNF-R1. In addition, the N-terminal region of TRUSS (residues 1 to 440) contains sequences that permit association with the cytoplasmic domain of TNF-R1. Transient overexpression of TRUSS activated NF-kappaB and increased NF-kappaB activation in response to ligation of TNF-R1. In contrast, a COOH-terminal-deletion mutant of TRUSS (TRUSS(1-723)) was found to inhibit NF-kappaB activation by TNF-alpha. Co-precipitation and co-immunoprecipitation assays revealed that TRUSS can interact with TRADD, TRAF2, and components of the IKK complex. These findings suggest that TRUSS may serve as a scaffolding protein that interacts with TNF-R1 signaling proteins and may link TNF-R1 to the activation of IKK.

Peroxisome Proliferator-active Receptor Gamma: a Legitimate Target to Control Pulmonary Inflammation?

American Journal of Respiratory and Critical Care Medicine. Jan, 2004  |  Pubmed ID: 14718232

Differential Regulation of TNF-R1 Signaling: Lipid Raft Dependency of P42mapk/erk2 Activation, but Not NF-kappaB Activation

Journal of Immunology (Baltimore, Md. : 1950). Jun, 2004  |  Pubmed ID: 15187147

The TNFR, TNF-R1, is localized to lipid raft and nonraft regions of the plasma membrane. Ligand binding sets in motion signaling cascades that promote the activation of p42(mapk/erk2) and NF-kappaB. However, the role of receptor localization in the activation of downstream signaling events is poorly understood. In this study, we investigated the dynamics of TNF-R1 localization to lipid rafts and the consequences of raft localization on the activation of p42(mapk/erk2) and NF-kappaB in primary cultures of mouse macrophages. Using sucrose density gradient ultracentrifugation and a sensitive ELISA to detect TNF-R1, we show that TNF-R1 is rapidly and transiently recruited to lipid rafts in response to TNF-alpha. Disruption of lipid rafts by cholesterol depletion prevented the TNF-alpha-dependent recruitment of TNF-R1 to lipid rafts and inhibited the activation of p42(mapk/erk2), while the activation of NF-kappaB was unaffected. In addition, phosphorylated p42(mapk/erk2), but not receptor interacting protein, I-kappaB kinase-gamma, or I-kappaBalpha was detected in raft-containing fractions following TNF-alpha stimulation. These findings suggest that TNF-R1 is localized to both lipid raft and nonraft regions of the plasma membrane and that each compartment is capable of initiating different signaling responses. We propose that segregation of TNF-R1 to raft and nonraft regions of the plasma membrane contributes to the diversity of signaling responses initiated by TNF-R1.

IL-4-induced Macrophage-derived IGF-I Protects Myofibroblasts from Apoptosis Following Growth Factor Withdrawal

Journal of Leukocyte Biology. Nov, 2004  |  Pubmed ID: 15316031

The development of idiopathic pulmonary fibrosis (IPF) is associated with myofibroblast accumulation and collagen deposition in the lung parenchyma. Recent studies have suggested that the fibroproliferative response is associated with immune deviation toward a T helper cell type 2 (Th2) cytokine profile. In addition, myofibroblast accumulation may be the result of resistance to physiologic apoptosis. If and how these events are linked remain largely unknown. Insulin-like growth factor-I (IGF-I) is a fibroblast growth and survival factor that has long been implicated in the pathogenesis of IPF. We have previously shown that interstitial macrophage-derived IGF-I correlates with disease severity in IPF, and the Th2 cytokines interleukin (IL)-4 and IL-13 stimulate the expression and secretion of IGF-I by macrophages. In the present study, we tested the hypothesis that IL-4-induced, macrophage-derived IGF-I protects myofibroblasts from apoptosis. Using a growth factor withdrawal model of apoptosis in the myofibroblast cell line, CCL39, we demonstrate that conditioned media from IL-4-stimulated macrophages protect myofibroblasts from apoptosis. The survival effect is lost when IGF-I is immunodepleted from macrophage-conditioned media with IGF-I-specific antibodies. We also show that the protection of myofibroblasts by macrophage-derived IGF-I correlates with and is dependent on the activation of the prosurvival kinases Akt and extracellular signal-regulated kinase. These findings support the view that IL-4-stimulated, macrophage-derived IGF-I may contribute to the persistence of myofibroblasts in pulmonary fibrosis in the Th2-deviated environment of the fibrotic lung.

Phosphorylation of Tumor Necrosis Factor Receptor 1 (p55) Protects Macrophages from Silica-induced Apoptosis

The Journal of Biological Chemistry. Jan, 2004  |  Pubmed ID: 14570868

Macrophages play a fundamental role in silicosis in part by removing silica particles and producing inflammatory mediators in response to silica. Tumor necrosis factor alpha (TNFalpha) is a prominent mediator in silicosis. Silica induction of apoptosis in macrophages might be mediated by TNFalpha. However, TNFalpha also activates signal transduction pathways (NF-kappaB and AP-1) that rescue cells from apoptosis. Therefore, we studied the TNFalpha-mediated mechanisms that confer macrophage protection against the pro-apoptotic effects of silica. We will show that exposure to silica induced TNFalpha production by RAW 264.7 cells, but not by IC-21. Silica-induced activation of NF-kappaB and AP-1 was only observed in RAW 264.7 macrophages. ERK activation in response to silica exposure was only observed in RAW 264.7 macrophages, whereas activation of p38 phosphorylation was predominantly observed in IC-21 macrophages. No changes in JNK activity were observed in either cell line in response to silica exposure. Silica induced apoptosis in both macrophage cell lines, but the induction of apoptosis was significantly larger in IC-21 cells. Protection against apoptosis in RAW 264.7 cells in response to silica was mediated by enhanced NF-kappaB activation and ERK-mediated phosphorylation of the p55 TNFalpha receptor. Inhibition of these two protective mechanisms by specific pharmacological inhibitors or transfection of dominant negative mutants that inhibit IkappaBalpha or ERK phosphorylation significantly increased silica-induced apoptosis in RAW 264.7 macrophages. These data suggest that NF-kappaB activation and ERK-mediated phosphorylation of the p55 TNF receptor are important cell survival mechanisms in the macrophage response to silica exposure.

Human Insulin-like Growth Factor-IA Expression in Transgenic Mice Promotes Adenomatous Hyperplasia but Not Pulmonary Fibrosis

American Journal of Physiology. Lung Cellular and Molecular Physiology. May, 2005  |  Pubmed ID: 15618451

Insulin-like growth factor-I (IGF-I) has been implicated in postnatal alveolar development, pulmonary fibrosis, and non-small cell lung cancer. To further investigate the role of IGF-I, we created a line of transgenic mice in which alveolar type II epithelial cells express human IGF-IA under the control of the surfactant protein C promoter. We determined the effect of pulmonary overexpression of human IGF-IA on 1) pulmonary inflammation and fibrosis in response to intratracheal instillation of bleomycin, 2) premalignant pulmonary adenomatous hyperplasia, and 3) adenoma formation. Transgenic expression of human IGF-IA had no effect on baseline gross lung pathology, cellularity of bronchoalveolar lavage, or total lung collagen content. In addition, there were no significant differences between transgenic mice and nontransgenic littermate controls in the development of pulmonary inflammation or pulmonary fibrosis in response to intratracheal bleomycin instillation. However, pulmonary expression of human IGF-IA in older mice (>12 mo) significantly increased the incidence of premalignant adenomatous hyperplastic lesions compared with littermate controls without affecting adenoma formation. These findings suggest that increased expression of human IGF-IA in alveolar air spaces does not affect the development of pulmonary fibrosis but promotes premalignant changes in the alveolar epithelium.

Transcription of Macrophage IGF-I Exon 1 is Positively Regulated by the 5'-untranslated Region and Negatively Regulated by the 5'-flanking Region

American Journal of Physiology. Lung Cellular and Molecular Physiology. Jun, 2005  |  Pubmed ID: 15681396

Idiopathic pulmonary fibrosis (IPF) is an insidious lung disease with no known cure or effective therapy. Macrophage-derived insulin-like growth factor-I (IGF-I) is thought to play a role in the pathogenesis of IPF; however, little is known about the control of IGF-I expression in macrophages. In this report we investigated the cis-regulatory elements that control basal expression using luciferase reporter constructs in RAW 264.7 macrophages. We show that the +95 to +329 region contains elements necessary to direct maximal promoter activity, whereas the +251 to +329 region contains the minimal promoter. Mapping transcriptional start sites for endogenous IGF-I in primary macrophages revealed that the major transcriptional start site is centered at +150, whereas the most 3'-transcriptional start site is centered at +255. Nuclear proteins from primary and RAW 264.7 macrophages bind specifically to the region required for maximal promoter activity (+134 to +173) and to the region required for minimal promoter activity (+267 to +299). Antibody supershift assays indicate that Sp3 bound to the +267 to +299 region. Moreover, mutation of the putative binding site reduced Sp3 binding in EMSAs and increased promoter activity in luciferase reporter gene assays. We also found that the regions from -1711 to -855 and -855 to -337 contain putative macrophage-specific suppressor elements that do not function in HeLa or COS-7 epithelial cell lines. These data support the view that macrophage IGF-I expression is positively regulated by elements located in the 5'-untranslated region and negatively regulated by elements in the 5'-flanking region of the IGF-I gene.

Hierarchical Phosphorylation of the TNF-alpha Receptor, TNF-R1, by P42Mapk/Erk at Basic Pro-directed Kinase Sites

Biochemistry. May, 2005  |  Pubmed ID: 15865443

Phosphorylation of the TNF-alpha receptor TNF-R1 has been shown to differentially regulate receptor signaling and function and promote changes in its subcellular localization. Previous studies have shown that p42(mapk/erk2) phosphorylates Ser and Thr residues (T236, S240, S244, and S270) in the membrane proximal region of TNF-R1 and that mutation of these residues to Glu and Asp residues (TNF-R1.4D/E) mimics the effect of phosphorylation on receptor signaling and localization. In the present study, we investigated whether the initial phosphorylation of these residues by p42(mapk/erk2) promotes hierarchical phosphorylation of additional sites within the cytoplasmic domain of TNF-R1. This question was addressed by investigating the ability of the TNF-R1.4D/E mutant receptor to be phosphorylated in in vitro kinase assays using GST-mutant cytoplasmic domain fusion proteins as substrates and in intact cells following mutant receptor expression. In addition, we determined the location of the additional phosphorylation sites. Incubation of Sepharose bead-bound GST-TNF-R1(207)(-)(425).4D/E fusion protein with lysates containing activated p42(mapk/erk2) led to the phosphorylation of Ser and Thr residues in addition to the previously defined sites at T236, S240, S244, and S270. Deletional mutagenesis localized these residues to a stretch of 14 amino acids that encompasses three basic Pro-directed ([S/T]P) kinase consensus sequences located between residues S256 and T267. Point mutagenesis of T257, S262, and T267 to Ala residues indicated that these sites are targets of phosphorylation by p42(mapk/)(erk2). These findings support the conclusion that p42(mapk/erk2) promotes extensive phosphorylation of the membrane proximal region in a hierarchical fashion at both consensus and nonconsensus ERK-phosphorylation sites.

ERK-mediated Phosphorylation of Thr735 in TNFalpha-converting Enzyme and Its Potential Role in TACE Protein Trafficking

Journal of Cell Science. Jun, 2005  |  Pubmed ID: 15923650

Tumor necrosis factor alpha-converting enzyme (TACE/ADAM17) has been implicated in the inducible shedding of numerous cell surface proteins. In light of this, the regulation of TACE catalytic activation and protein maturation has recently received considerable interest in the context of extracellular signal-regulated kinase activation and the subsequent phosphorylation of TACE at residue Thr735. In this study, we analysed the subcellular localisation of TACE derivatives using laser-scanning confocal microscopy and cell surface biotinylation. Whereas WT.TACE- and T735A.TACE-enhanced green fluorescent protein (-eGFP) fusion derivatives of TACE were both found to localise with the endoplasmic reticulum, a phosphomimicking form of TACE (T735E.TACE-eGFP) was found to colocalise predominantly with components of the protein secretory pathway (COPII vesicles and trans-Golgi network). Additionally, upon analysis of biotinylated ectopic TACE, we observed that inducible trafficking of TACE to the cell surface was dependent upon extracellular signal-regulated kinase activation and phosphorylation of Thr735. We conclude from our observations that phosphorylation of TACE at Thr735 highlights a key step in inducible TACE protein trafficking and maturation.

TNF-alpha Sensitizes Normal and Fibrotic Human Lung Fibroblasts to Fas-induced Apoptosis

American Journal of Respiratory Cell and Molecular Biology. Mar, 2006  |  Pubmed ID: 16272460

Pulmonary accumulation of fibroblasts and myofibroblasts in idiopathic pulmonary fibrosis/usual interstitial pneumonia (IFP/UIP) has been linked to (1) increased migration of a circulating pool of fibrocytes, (2) cell proliferation, and (3) resistance to apoptosis. The mechanism of physiologic apoptosis of lung fibroblasts is poorly understood. Using normal and fibrotic human lung fibroblasts and the human lung fibroblast cell line, MRC-5, we examined the regulation of Fas-induced apoptosis by the proinflammatory cytokines TNF-alpha and IFN-gamma. Herein, we show that the basal resistance of lung fibroblasts and myofibroblasts to Fas-induced apoptosis is overcome by sensitization with TNF-alpha. IFN-gamma did not sensitize cells to Fas-induced apoptosis, but exhibited synergistic activity with TNF-alpha. Sensitization by TNF-alpha was observed in MRC-5 cells and in fibroblasts and myofibroblasts from normal and fibrotic human lung, suggesting that this represents a conserved mechanism to engage Fas-induced apoptosis. The mechanism of sensitization was localized at the level of recruitment of the adapter protein, FADD, to the cytoplasmic domain of Fas. Collectively, these findings suggest that fibroblast apoptosis involves two steps, sensitization and induction, and that inadequate pulmonary inflammation in IPF/UIP may favor fibroblast accumulation by reducing sensitization to apoptosis.

Regulation of Chemokine Expression by NaCl Occurs Independently of Cystic Fibrosis Transmembrane Conductance Regulator in Macrophages

The American Journal of Pathology. Jul, 2006  |  Pubmed ID: 16816357

Chronic pulmonary inflammation and infection are the leading causes of morbidity and mortality in cystic fibrosis (CF). While the effect of mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) on airways remains controversial, some groups have demonstrated increases in Na(+) and Cl(-) in CF airway surface liquid compared to normal airways. We investigated the consequences of NaCl on pro-inflammatory chemokine and cytokine production by macrophages. Stimulation of mouse macrophages with increasing amounts of NaCl induced macrophage inflammatory protein-2 (MIP-2) and tumor necrosis factor-alpha (TNF-alpha) production. Further, co-incubation of macrophages with NaCl in the presence of either lipopolysaccharide (LPS) or TNF-alpha synergistically increased MIP-2 production. Both the NaCl and NaCl plus LPS responses were partially dependent on endogenous production and autocrine signaling by TNF-alpha. To investigate the role of CFTR in MIP-2 production, we compared the responses of wild-type and DeltaF508 CF mouse macrophages to NaCl and LPS. The responses of macrophages from both strains were indistinguishable. In addition, CFTR mRNA was not expressed in macrophages. Taken together, these findings suggest that NaCl stimulates MIP-2 production by macrophages through a mechanism that is partially dependent on TNF-alpha but independent of macrophage CFTR expression.

TRUSS, a Tumor Necrosis Factor Receptor-1-interacting Protein, Activates C-Jun NH(2)-terminal Kinase and Transcription Factor AP-1

FEBS Letters. Aug, 2006  |  Pubmed ID: 16876162

Tumor necrosis factor-alpha (TNF-alpha) induces the transcriptional activation of numerous genes involved in the inflammatory response. The recently identified protein TRUSS was investigated for its role in TNF-alpha-induced activation of c-Jun-NH(2) terminal kinase (JNK) and transcription factor, AP-1. Ectopic expression of TRUSS activated JNK and AP-1 in the absence and presence of TNF-alpha stimulation. The C-terminal region of TRUSS interacted with TNF receptor-associated factor-2 (TRAF2) and co-expression of dominant-inhibitory TRAF2 with TRUSS inhibited AP-1 activation, suggesting that TRUSS signaling occurs upstream of TRAF2. Further, a dominant-inhibitory mutant of TRUSS inhibited TNF-alpha-induced AP-1 activation. These findings suggest that TRUSS activates JNK in a TRAF2-dependent fashion and is involved in TNF-alpha-induced AP-1 activation via JNK kinases.

Symposium: Thomas L. Petty Aspen Lung Conference: Lung Injury and Repair. Introduction and Perspective

Proceedings of the American Thoracic Society. Apr, 2008  |  Pubmed ID: 18403319

Corticosteroid-resistant Asthma is Associated with Classical Antimicrobial Activation of Airway Macrophages

The Journal of Allergy and Clinical Immunology. Sep, 2008  |  Pubmed ID: 18774390

The cause of corticosteroid-resistant (CR) asthma is unknown.

Impaired Apoptotic Cell Clearance in CGD Due to Altered Macrophage Programming is Reversed by Phosphatidylserine-dependent Production of IL-4

Blood. Feb, 2009  |  Pubmed ID: 18952895

Chronic granulomatous disease (CGD) is characterized by overexuberant inflammation and autoimmunity that are attributed to deficient anti-inflammatory signaling. Although regulation of these processes is complex, phosphatidylserine (PS)-dependent recognition and removal of apoptotic cells (efferocytosis) by phagocytes are potently anti-inflammatory. Since macrophage phenotype also plays a beneficial role in resolution of inflammation, we hypothesized that impaired efferocytosis in CGD due to macrophage skewing contributes to enhanced inflammation. Here we demonstrate that efferocytosis by macrophages from CGD (gp91(phox)(-/-)) mice was suppressed ex vivo and in vivo. Alternative activation with interleukin 4 (IL-4) normalized CGD macrophage efferocytosis, whereas classical activation by lipopolysaccharide (LPS) plus interferon gamma (IFNgamma) had no effect. Importantly, neutralization of IL-4 in wild-type macrophages reduced macrophage efferocytosis, demonstrating a central role for IL-4. This effect was shown to involve 12/15 lipoxygenase and activation of peroxisome-proliferator activated receptor gamma (PPARgamma). Finally, injection of PS (whose exposure is lacking on CGD apoptotic neutrophils) in vivo restored IL-4-dependent macrophage reprogramming and efferocytosis via a similar mechanism. Taken together, these findings support the hypothesis that impaired PS exposure on dying cells results in defective macrophage programming, with consequent efferocytic impairment and has important implications in understanding the underlying cause of enhanced inflammation in CGD.

Pressure-mediated Hypertrophy and Mechanical Stretch Induces IL-1 Release and Subsequent IGF-1 Generation to Maintain Compensative Hypertrophy by Affecting Akt and JNK Pathways

Circulation Research. Nov, 2009  |  Pubmed ID: 19834007

It has been reported that interleukin (IL)-1 is associated with pathological cardiac remodeling and LV dilatation, whereas IL-1beta has also been shown to induce cardiomyocyte hypertrophy. Thus, the role of IL-1 in the heart remains to be determined.

Lung Tumor Growth is Stimulated in IFN-gamma-/- Mice and Inhibited in IL-4Ralpha-/- Mice

Anticancer Research. Dec, 2009  |  Pubmed ID: 20044622

Alternative (M2) macrophage activation is associated with tumor development in many tumor types, including those in the lung. Herein the biological consequences of forcing classical (M1) or alternative (M2) macrophage activation on lung tumor development are examined.

TRUSS, TNF-R1, and TRPC Ion Channels Synergistically Reverse Endoplasmic Reticulum Ca2+ Storage Reduction in Response to M1 Muscarinic Acetylcholine Receptor Signaling

Journal of Cellular Physiology. Nov, 2010  |  Pubmed ID: 20458742

Although most signaling responses initiated by tumor necrosis factor-alpha (TNF-alpha) occur in a Ca(2+)-independent fashion, TNF-alpha receptor signaling augments Ca(2+) entry induced by Galpha(q/11) G-protein coupled receptors (GPCRs) in endothelial cells and increases trans-endothelial permeability. The signaling events involved in GPCR-induced Ca(2+) influx have been characterized and involve store-operated Ca(2+) entry facilitated by the Ca(2+) permeable ion channel, transient receptor potential canonical 4 (TRPC4). Little is known about the mechanisms by which TNF-alpha receptor signaling augments GPCR-induced Ca(2+) entry. TNF-alpha Receptor Ubiquitous Signaling and Scaffolding protein (TRUSS) is a tumor necrosis factor receptor-1 (TNF-R1)-associated protein whose gene name is TRPC4-associated protein (TRPC4AP). The goal of our study was to test the hypothesis that TRUSS serves to link TNF-R1 and GPCR-signaling pathways at the level of TRPC4 by: (i) determining if TRUSS and TNF-R1 interact with TRPC4, and (ii) investigating the role of TRUSS, TNF-R1, and TRPC4 in GPCR-induced Ca(2+) signaling. Here, we show that TRUSS and TNF-R1 interact with a sub-family of TRPC channels (TRPC1, 4, and 5). In addition, we show that TRUSS and TNF-R1 function together with TRPC4 to elevate endoplasmic reticulum Ca(2+) filling in the context of reduced endoplasmic reticulum Ca(2+) storage initiated by G-protein coupled m1 muscarinic acetylcholine receptor (m1AchR) signaling. Together, these findings suggest that TNF-R1, TRUSS, and TRPC4 augment Ca(2+) loading of endoplasmic reticulum Ca(2+) stores in the context of m1AchR stimulation and provide new insights into the mechanisms that connect TNF-R1 to GPCR-induced Ca(2+) signaling.

Schistosomiasis-induced Experimental Pulmonary Hypertension: Role of Interleukin-13 Signaling

The American Journal of Pathology. Sep, 2010  |  Pubmed ID: 20671265

The mechanisms underlying schistosomiasis-induced pulmonary hypertension (PH), one of the most common causes of PH worldwide, remain unclear. We sought to determine whether Schistosoma mansoni causes experimental PH associated with pulmonary vascular remodeling in an interleukin (IL)-13-dependent manner. IL-13Ralpha1 is the canonical IL-13 signaling receptor, whereas IL-13Ralpha2 is a competitive nonsignaling decoy receptor. Wild-type, IL-13Ralpha1(-/-), and IL-13Ralpha2(-/-) C57BL/6J mice were percutaneously infected with S. mansoni cercariae, followed by i.v. injection of eggs. We assessed PH with right ventricular catheterization, histological evaluation of pulmonary vascular remodeling, and detection of IL-13 and transforming growth factor-beta signaling. Infected mice developed pulmonary peri-egg granulomas and arterial remodeling involving predominantly the vascular media. In addition, gain-of-function IL-13Ralpha2(-/-) mice had exacerbated vascular remodeling and PH. Mice with loss of IL-13Ralpha1 function did not develop PH and had reduced pulmonary vascular remodeling. Moreover, the expression of resistin-like molecule-alpha, a target of IL-13 signaling, was increased in infected wild-type and IL-13Ralpha2(-/-) but not IL-13Ralpha1(-/-) mice. Phosphorylated Smad2/3, a target of transforming growth factor-beta signaling, was increased in both infected mice and humans with the disease. Our data indicate that experimental schistosomiasis causes PH and potentially relies on up-regulated IL-13 signaling.

PPARγ Activation Normalizes Resolution of Acute Sterile Inflammation in Murine Chronic Granulomatous Disease

Blood. Nov, 2010  |  Pubmed ID: 20693431

Absence of a functional nicotinamide adenine dinucleotide phosphate (NADPH) oxidase predisposes chronic granulomatous disease (CGD) patients to infection, and also to unexplained, exaggerated inflammation. The impaired recognition and removal (efferocytosis) of apoptotic neutrophils by CGD macrophages may contribute to this effect. We hypothesized that peroxisome proliferator-activated receptor γ (PPARγ) activation during CGD inflammation is deficient, leading to altered macrophage programming and decreased efferocytosis, and that PPARγ agonism would enhance resolution. using the gp91(phox-/-) murine model of X-linked CGD in a well-characterized model of sterile, zymosan-induced peritonitis, it was demonstrated that PPARγ expression and activation in CGD macrophages were significantly deficient at baseline, and acquisition was delayed over the course of inflammation relative to that of wild-type. Efferocytosis by macrophages reflected PPARγ activation during peritonitis and was impaired in CGD mice (versus wild-type), leading to accumulation of apoptotic neutrophils. Importantly, provision of the PPARγ agonist, pioglitazone, either prophylactically or during inflammation, significantly enhanced macrophage PPARγ-mediated programming and efferocytosis, reduced accumulation of apoptotic neutrophils, and normalized the course of peritonitis in CGD mice. As such, PPARγ may be a therapeutic target for CGD, and possibly other inflammatory conditions where aberrant macrophage programming and impaired efferocytosis delay resolution of inflammation.

TNF Receptor-1 (TNF-R1) Ubiquitous Scaffolding and Signaling Protein Interacts with TNF-R1 and TRAF2 Via an N-terminal Docking Interface

Biochemistry. Sep, 2010  |  Pubmed ID: 20704259

TNF receptor-1 (TNF-R1) signal transduction is mediated through the assembly of scaffolding proteins, adaptors, and kinases. TNF receptor ubiquitous scaffolding and signaling protein (TRUSS), a 90.1 kDa TNF-R1-associated scaffolding protein, also interacts with TRAF2 and IKK and contributes to TNF-alpha-induced nuclear factor-kappaB (NF-kappaB) and c-Jun-NH(2)-terminal kinase (JNK) activation. Little is known about the mechanism of interaction among TRUSS, TNF-R1, and TRAF2. To address this issue, we used deletional and site-directed mutagenesis approaches to systematically investigate (i) the regions of TRUSS that interact with TNF-R1 and TRAF2 and (ii) the ability of TRUSS to self-associate to form higher-order complexes. Here we show that sequences located in the N-terminal (residues 1-248) and central (residues 249-440) regions of TRUSS are required to form a docking interface that supports binding to both TNF-R1 and TRAF2. While the C-terminal region (residues 441-797) did not directly interact with TNF-R1 or TRAF2, sequences located in this region were capable of self-association. Collectively, these data suggest that (i) the interaction between TNF-R1 and TRAF2 requires sequences located in the entire N-terminal half (residues 1-440) of TRUSS, (ii) the binding interface for TNF-R1 is closely linked with the TRAF2 binding interface, and (iii) the assembly of homomeric TRUSS complexes may contribute to its role in TNF-R1 signaling.

Impaired Phagocytosis of Apoptotic Cells by Macrophages in Chronic Granulomatous Disease is Reversed by IFN-γ in a Nitric Oxide-dependent Manner

Journal of Immunology (Baltimore, Md. : 1950). Oct, 2010  |  Pubmed ID: 20805415

Immunodeficiency in chronic granulomatous disease (CGD) is well characterized. Less understood are exaggerated sterile inflammation and autoimmunity associated with CGD. Impaired recognition and clearance of apoptotic cells resulting in their disintegration may contribute to CGD inflammation. We hypothesized that priming of macrophages (Ms) with IFN-γ would enhance impaired engulfment of apoptotic cells in CGD. Diverse M populations from CGD (gp91(phox)(-/-)) and wild-type mice, as well as human Ms differentiated from monocytes and promyelocytic leukemia PLB-985 cells (with and without mutation of the gp91(phox)), demonstrated enhanced engulfment of apoptotic cells in response to IFN-γ priming. Priming with IFN-γ was also associated with increased uptake of Ig-opsonized targets, latex beads, and fluid phase markers, and it was accompanied by activation of the Rho GTPase Rac. Enhanced Rac activation and phagocytosis following IFN-γ priming were dependent on NO production via inducible NO synthase and activation of protein kinase G. Notably, endogenous production of TNF-α in response to IFN-γ priming was critically required for inducible NO synthase upregulation, NO production, Rac activation, and enhanced phagocytosis. Treatment of CGD mice with IFN-γ also enhanced uptake of apoptotic cells by M in vivo via the signaling pathway. Importantly, during acute sterile peritonitis, IFN-γ treatment reduced excess accumulation of apoptotic neutrophils and enhanced phagocytosis by CGD Ms. These data support the hypothesis that in addition to correcting immunodeficiency in CGD, IFN-γ priming of Ms restores clearance of apoptotic cells and may thereby contribute to resolution of exaggerated CGD inflammation.

Compartmentalized Expression of C-FLIP in Lung Tissues of Patients with Idiopathic Pulmonary Fibrosis

American Journal of Respiratory Cell and Molecular Biology. Feb, 2010  |  Pubmed ID: 19372246

Increased apoptosis of alveolar epithelial cells and impaired apoptosis of myofibroblasts have been linked to the pathogenesis of idiopathic pulmonary fibrosis/usual interstitial pneumonia (IPF/UIP). Fas, a death receptor of the TNF-receptor superfamily, has been implicated in apoptosis of both cell types, though the mechanisms are poorly understood. The goals of this study were: (1) to examine the localization of Fas-associated death-domain-like IL-1beta-converting enzyme inhibitory protein (c-FLIP), an NF-kappaB-dependent regulator of Fas-signaling, in lung tissues from IPF/UIP patients and control subjects; and (2) to compare c-FLIP expression with epithelial cell and myofibroblast apoptosis, proliferation, and NF-kappaB activation. c-FLIP expression was restricted to airway epithelial cells in control lung tissues. In contrast, in patients with IPF/UIP, c-FLIP was also expressed by alveolar epithelial cells in areas of injury and fibrosis, but was absent from myofibroblasts in fibroblastic foci and from alveolar epithelial cells in uninvolved areas of lung tissue. Quantification of apoptosis and proliferation revealed an absence of apoptotic or proliferating cells in fibroblastic foci and low levels of apoptosis and proliferation by alveolar epithelial cells. Quantification of NF-kappaB expression and nuclear translocation revealed strong staining and translocation in alveolar epithelial cells and weak staining and minimal nuclear translocation in myofibroblasts. These findings suggest that: (1) c-FLIP expression is induced in the abnormal alveolar epithelium of patients with IPF/UIP, (2) the resistance of myofibroblasts to apoptosis in patients with IPF/UIP occurs independently of c-FLIP expression, and (3) increased NF-kappaB activation and c-FLIP expression by the alveolar epithelium may be linked.

Increased Cell Surface Fas Expression is Necessary and Sufficient to Sensitize Lung Fibroblasts to Fas Ligation-induced Apoptosis: Implications for Fibroblast Accumulation in Idiopathic Pulmonary Fibrosis

Journal of Immunology (Baltimore, Md. : 1950). Jul, 2011  |  Pubmed ID: 21632719

Idiopathic pulmonary fibrosis (IPF) is associated with the accumulation of collagen-secreting fibroblasts and myofibroblasts in the lung parenchyma. Many mechanisms contribute to their accumulation, including resistance to apoptosis. In previous work, we showed that exposure to the proinflammatory cytokines TNF-α and IFN-γ reverses the resistance of lung fibroblasts to apoptosis. In this study, we investigate the underlying mechanisms. Based on an interrogation of the transcriptomes of unstimulated and TNF-α- and IFN-γ-stimulated primary lung fibroblasts and the lung fibroblast cell line MRC5, we show that among Fas-signaling pathway molecules, Fas expression was increased ∼6-fold in an NF-κB- and p38(mapk)-dependent fashion. Prevention of the increase in Fas expression using Fas small interfering RNAs blocked the ability of TNF-α and IFN-γ to sensitize fibroblasts to Fas ligation-induced apoptosis, whereas enforced adenovirus-mediated Fas overexpression was sufficient to overcome basal resistance to Fas-induced apoptosis. Examination of lung tissues from IPF patients revealed low to absent staining of Fas in fibroblastic cells of fibroblast foci. Collectively, these findings suggest that increased expression of Fas is necessary and sufficient to overcome the resistance of lung fibroblasts to Fas-induced apoptosis. Our findings also suggest that approaches aimed at increasing Fas expression by lung fibroblasts and myofibroblasts may be therapeutically relevant in IPF.

Age and Sex Dimorphisms Contribute to the Severity of Bleomycin-induced Lung Injury and Fibrosis

American Journal of Physiology. Lung Cellular and Molecular Physiology. Oct, 2011  |  Pubmed ID: 21743030

Fibrotic interstitial pneumonias are more prevalent in males of advancing age, although little is known about the underlying mechanisms. To evaluate the contributions of age and sex to the development of pulmonary fibrosis, we intratracheally instilled young (8-12 wk) and aged (52-54 wk) male and female mice with bleomycin and assessed the development and severity of fibrotic lung disease by measurements of lung collagen levels, static compliance, leukocyte infiltration, and stereological quantification of fibrotic areas in histological sections. We also quantified proinflammatory and profibrotic chemokine and cytokine levels in the bronchoalveolar lavage fluid. Aged male mice developed more severe lung disease, indicated by increased mortality, increased collagen deposition, and neutrophilic alveolitis compared with aged female mice or young mice of either sex. Aged male mice also exhibited increased levels of transforming growth factor-β, IL-17A, and CXCL1 in their bronchoalveolar lavage fluid. Young male mice developed a more fibrotic disease after bleomycin instillation compared with female mice, regardless of age. There was no difference in fibrosis between young and aged female mice. Taken together, these findings suggest that the variables of advanced age and male sex contribute to the severity of pulmonary fibrosis in this model. Our findings also emphasize the importance of stratifying experimental groups on the basis of age and sex in experimental and epidemiological studies of this nature.

Lung Dendritic Cells at the Innate-adaptive Immune Interface

Journal of Leukocyte Biology. Nov, 2011  |  Pubmed ID: 21807741

This review updates the basic biology of lung DCs and their functions. Lung DCs have taken center stage as cellular therapeutic targets in new vaccine strategies for the treatment of diverse human disorders, including asthma, allergic lung inflammation, lung cancer, and infectious lung disease. The anatomical distribution of lung DCs, as well as the division of labor between their subsets, aids their ability to recognize and endocytose foreign substances and to process antigens. DCs can induce tolerance in or activate naïve T cells, making lung DCs well-suited to their role as lung sentinels. Lung DCs serve as a functional signaling/sensing unit to maintain lung homeostasis and orchestrate host responses to benign and harmful foreign substances.

Matrix Metalloproteinase 3 is a Mediator of Pulmonary Fibrosis

The American Journal of Pathology. Oct, 2011  |  Pubmed ID: 21871427

Idiopathic pulmonary fibrosis (IPF) may be triggered by epithelial injury that results in aberrant production of growth factors, cytokines, and proteinases, leading to proliferation of myofibroblasts, excess deposition of collagen, and destruction of the lung architecture. The precise mechanisms and key signaling mediators responsible for this aberrant repair process remain unclear. We assessed the importance of matrix metalloproteinase-3 (MMP-3) in the pathogenesis of IPF through i) determination of MMP-3 expression in patients with IPF, ii) in vivo experiments examining the relevance of MMP-3 in experimental models of fibrosis, and iii) in vitro experiments to elucidate possible mechanisms of action. Gene expression analysis, quantitative RT-PCR, and Western blot analysis of explanted human lungs revealed enhanced expression of MMP-3 in IPF, compared with control. Transient adenoviral vector-mediated expression of recombinant MMP-3 in rat lung resulted in accumulation of myofibroblasts and pulmonary fibrosis. Conversely, MMP-3-null mice were protected against bleomycin-induced pulmonary fibrosis. In vitro treatment of cultured lung epithelial cells with purified MMP-3 resulted in activation of the β-catenin signaling pathway, via cleavage of E-cadherin, and induction of epithelial-mesenchymal transition. These processes were inhibited in bleomycin-treated MMP-3-null mice, as assessed by cytosolic translocation of β-catenin and cyclin D1 expression. These observations support a novel role for MMP-3 in the pathogenesis of IPF, through activation of β-catenin signaling and induction of epithelial-mesenchymal transition.

A Novel Model of Rheumatoid Arthritis-associated Interstitial Lung Disease in SKG Mice

Experimental Lung Research. Mar, 2012  |  Pubmed ID: 22185348

ABSTRACT Rheumatoid arthritis-associated interstitial lung disease (RA-ILD) is associated with increased mortality in up to 10% of patients with rheumatoid arthritis. Lung exposure to cigarette smoke has been implicated in disease development. Little is known about the mechanisms underlying the development of RA-ILD, in part due to the lack of an appropriate mouse model. The objectives of this study were (i) to test the suitability of SKG mice as a model of cellular and fibrotic interstitial pneumonia in the setting of autoimmune arthritis, and (ii) to determine the role of lung injury in the development of arthritis in SKG mice. Lung tissues were evaluated in arthritic SKG mice by quantifying cell accumulation in bronchoalveolar lavage, static compliance, collagen levels, and infiltrating cell phenotypes by flow cytometry and histology. Lung injury was induced by exposure to cigarette smoke or bleomycin. Arthritic SKG mice developed a patchy cellular and fibrotic interstitial pneumonia associated with reduced static compliance, increased collagen levels, and accumulation of inflammatory cells. Infiltrating cells comprised CD4(+) T cells, B cells, macrophages, and neutrophils. Chronic exposure to cigarette smoke or initiation of lung injury with bleomycin did not cause arthritis. The pattern of lung disease suggests that arthritic SKG mice represent an authentic model of nonspecific interstitial pneumonia in RA-ILD patients. The lack of arthritis development after cigarette smoke or lung injury suggests that a model where breaches in immunologic tolerance are induced by lung inflammation and injury alone may be overly simplistic.

Spatiotemporal Inhibition of Innate Immunity Signaling by the Tbc1d23 RAB-GAP

Journal of Immunology (Baltimore, Md. : 1950). Feb, 2012  |  Pubmed ID: 22312129

We previously identified Tbc1d23 as a candidate novel regulator of innate immunity using comparative genomics RNA interference screens in Caenorhabditis elegans and mouse macrophages. Using Tbc1d23 knockout mice and macrophages engineered to overexpress Tbc1d23, we now show that Tbc1d23 is a general inhibitor of innate immunity signaling, strongly inhibiting multiple TLR and dectin-signaling pathways. Tbc1d23 likely acts downstream of the TLR-signaling adaptors MyD88 and Trif and upstream of the transcription factor XBP1. Importantly, like XBP1, Tbc1d23 affects the maintenance, but not the initiation, of inflammatory cytokine production induced by LPS. Tbc1d23 acts as a RAB-GAP to regulate innate immunity signaling. Thus, Tbc1d23 exerts its inhibitory effect on innate immunity signaling in a spatiotemporal fashion. The identification of a novel spatiotemporal regulator of innate immunity signaling validates the comparative genomics approach for innate immunity gene discovery.